Individual DNAs isolated from four sublines of AKR mice (AKR/J, AKR/N, AKR/Cum, and AKR/Boy) have been examined by Southern-blot analysis following digestion with several restriction enzymes. A 500 base pair, 32p-labeled, cloned ecotropic murine leukemia virus (MuLV) envelope-specific probe was used to identify restriction fragments that contain ecotropic MuLV proviral sequences. Variation in the number of proviral copies and in their qenomic organization as reflected by the location of restriction enzyme sites in flanking cellular sequences was observed both between and within AKR sublines. The results obtained are consistent with a continual acquisition of new proviral copies in the four sublines studied. Ecotropic proviral DNA copies shared by the four AKR sublines are related to their geneology; each subline contains two or three copies of proviral DNA in common with other sublines and from one to six unique ecotropic proviruses. Some of the unique proviral DNA copies contain internal alterations as reflected by restriction enzyme maps that differ from those of prototype ecotropic provirus